Jodi L. Vogel

Inhibition of the histone demethylase LSD1 blocks α-herpesvirus lytic replication and reactivation from latency

Reversible methylation of histone tails serves as either a positive signal recognized by transcriptional assemblies or a negative signal that result in repression. Invading viral pathogens that depend upon the host cells transcriptional apparatus are also subject to the regulatory impact of chromatin assembly and modifications. Here we show that infection by the α-herpesviruses, herpes simplex virus (HSV) and varicella zoster virus (VZV), results in the rapid accumulation of chromatin bearing repressive histone H3 Lys9 methylation. To enable expression of viral immediate early (IE) genes, both viruses use the cellular transcriptional coactivator host cell factor-1 (HCF-1) to recruit the lysine-specific demethylase-1 (LSD1) to the viral immediate early promoters. Depletion of LSD1 or inhibition of its activity with monoamine oxidase inhibitors (MAOIs) results in the accumulation of repressive chromatin and a block to viral gene expression. As HCF-1 is a component of the Set1 and MLL1 histone H3 Lys4 methyltransferase complexes, it thus coordinates modulation of repressive H3 Lys9 methylation levels with addition of activating H3 Lys4 trimethylation marks. Strikingly, MAOIs also block the reactivation of HSV from latency in sensory neurons, indicating that the HCF-1 complex is a crucial component of the reactivation mechanism. The results support pharmaceutical control of histone modifying enzymes as a strategy for controlling herpesvirus infections.

Heat-shock proteins Hsp104 and Hsp70 reactivate mRNA splicing after heat inactivation.

BACKGROUND The heat-shock protein Hsp104 plays a crucial role in the survival of cells exposed to high temperatures and other severe stresses, but its specific functions and the biological pathways on which it operates have been unclear. Indeed, very little is known about the specific cellular processes in which any of the heat-shock proteins acts to affect thermotolerance. One essential process that is particularly sensitive to heat in many organisms is the splicing of intervening sequences from mRNA precursors. RESULTS We have examined the role of Hsp104 in the repair of splicing after disruption by heat shock. When splicing in the budding yeast Saccharomyces cerevisiae was disrupted by a brief heat shock, it recovered much more rapidly in wild-type strains than in strains containing hsp104 mutations. Constitutive expression of Hsp104 promoted the recovery of heat-damaged splicing in the absence of other protein synthesis, but did not protect splicing from the initial disruption, suggesting that Hsp104 functions to repair splicing after heat damage rather than to prevent the initial damage. A modest reduction in the recovery of splicing after heat shock in an hsp70 mutant suggested that Hsp70 may also function in the repair of splicing. The roles of Hsp104 and Hsp70 were confirmed by the ability of the purified proteins to restore splicing in extracts that had been heat-inactivated in vitro. Together, these two proteins were able to restore splicing to a greater degree than could be accomplished by an optimal concentration of either protein alone. CONCLUSIONS Our findings provide the first demonstration of the roles of heat-shock proteins in a biological process that is known to be particularly sensitive to heat in vivo. The results support previous genetic arguments that the Hsp104 and Hsp70 proteins have different, but related, functions in protecting cells from the toxic effects of high temperatures. Because Hsp104 and Hsp70 are able to function in vitro, after the heat-damaged substrate or substrates have been generated, neither protein is required to bind to its target(s) during heating in order to effect repair.

The novel coactivator C1 (HCF) coordinates multiprotein enhancer formation and mediates transcription activation by GABP.

Transcription of the herpes simplex virus 1 (HSV‐1) immediate early (IE) genes is determined by multiprotein enhancer complexes. The core enhancer assembly requires the interactions of the POU‐homeodomain protein Oct‐1, the viral transactivator αTIF and the cellular factor C1 (HCF). In this context, the C1 factor interacts with each protein to assemble the stable enhancer complex. In addition, the IE enhancer cores contain adjacent binding sites for other cellular transcription factors such as Sp1 and GA‐binding protein (GABP). In this study, a direct interaction of the C1 factor with GABP is demonstrated, defining the C1 factor as the critical coordinator of the enhancer complex assembly. In addition, mutations that reduce the GABP transactivation potential also impair the C1–GABP interaction, indicating that the C1 factor functions as a novel coactivator of GABP‐mediated transcription. The interaction and coordinated assembly of the enhancer proteins by the C1 factor may be critical for the regulation of the HSV lytic–latent cycle.

A Novel Selective LSD1/KDM1A Inhibitor Epigenetically Blocks Herpes Simplex Virus Lytic Replication and Reactivation from Latency

ABSTRACT Cellular processes requiring access to the DNA genome are regulated by an overlay of epigenetic modifications, including histone modification and chromatin remodeling. Similar to the cellular host, many nuclear DNA viruses that depend upon the host cell’s transcriptional machinery are also subject to the regulatory impact of chromatin assembly and modification. Infection of cells with alphaherpesviruses (herpes simplex virus [HSV] and varicella-zoster virus [VZV]) results in the deposition of nucleosomes bearing repressive histone H3K9 methylation on the viral genome. This repressive state is modulated by the recruitment of a cellular coactivator complex containing the histone H3K9 demethylase LSD1 to the viral immediate-early (IE) gene promoters. Inhibition of the activity of this enzyme results in increased repressive chromatin assembly and suppression of viral gene expression during lytic infection as well as reactivation from latency in a mouse ganglion explant model. However, available small-molecule LSD1 inhibitors are not originally designed to inhibit LSD1, but rather monoamine oxidases (MAO) in general. Thus, their specificity for and potency to LSD1 is low. In this study, a novel specific LSD1 inhibitor was identified that potently repressed HSV IE gene expression, genome replication, and reactivation from latency. Importantly, the inhibitor also suppressed primary infection of HSV in vivo in a mouse model. Based on common control of a number of DNA viruses by epigenetic modulation, it was also demonstrated that this LSD1 inhibitor blocks initial gene expression of the human cytomegalovirus and adenovirus type 5. IMPORTANCE Epigenetic mechanisms, including histone modification and chromatin remodeling, play important regulatory roles in all cellular processes requiring access to the genome. These mechanisms are often altered in disease conditions, including various cancers, and thus represent novel targets for drugs. Similarly, many viral pathogens are regulated by an epigenetic overlay that determines the outcome of infection. Therefore, these epigenetic targets also represent novel antiviral targets. Here, a novel inhibitor was identified with high specificity and potency for the histone demethylase LSD1, a critical component of the herpes simplex virus (HSV) gene expression paradigm. This inhibitor was demonstrated to have potent antiviral potential in both cultured cells and animal models. Thus, in addition to clearly demonstrating the critical role of LSD1 in regulation of HSV infection, as well as other DNA viruses, the data extends the therapeutic potential of chromatin modulation inhibitors from the focused field of oncology to the arena of antiviral agents. Epigenetic mechanisms, including histone modification and chromatin remodeling, play important regulatory roles in all cellular processes requiring access to the genome. These mechanisms are often altered in disease conditions, including various cancers, and thus represent novel targets for drugs. Similarly, many viral pathogens are regulated by an epigenetic overlay that determines the outcome of infection. Therefore, these epigenetic targets also represent novel antiviral targets. Here, a novel inhibitor was identified with high specificity and potency for the histone demethylase LSD1, a critical component of the herpes simplex virus (HSV) gene expression paradigm. This inhibitor was demonstrated to have potent antiviral potential in both cultured cells and animal models. Thus, in addition to clearly demonstrating the critical role of LSD1 in regulation of HSV infection, as well as other DNA viruses, the data extends the therapeutic potential of chromatin modulation inhibitors from the focused field of oncology to the arena of antiviral agents.

The THAP-Zinc Finger Protein THAP1 Associates with Coactivator HCF-1 and O-GlcNAc Transferase A LINK BETWEEN DYT6 AND DYT3 DYSTONIAS

THAP1 is a sequence-specific DNA binding factor that regulates cell proliferation through modulation of target genes such as the cell cycle-specific gene RRM1. Mutations in the THAP1 DNA binding domain, an atypical zinc finger (THAP-zf), have recently been found to cause DYT6 dystonia, a neurological disease characterized by twisting movements and abnormal postures. In this study, we report that THAP1 shares sequence characteristics, in vivo expression patterns and protein partners with THAP3, another THAP-zf protein. Proteomic analyses identified HCF-1, a potent transcriptional coactivator and cell cycle regulator, and O-GlcNAc transferase (OGT), the enzyme that catalyzes the addition of O-GlcNAc, as major cellular partners of THAP3. THAP3 interacts with HCF-1 through a consensus HCF-1-binding motif (HBM), a motif that is also present in THAP1. Accordingly, THAP1 was found to bind HCF-1 in vitro and to associate with HCF-1 and OGT in vivo. THAP1 and THAP3 belong to a large family of HCF-1 binding factors since seven other members of the human THAP-zf protein family were identified, which harbor evolutionary conserved HBMs and bind to HCF-1. Chromatin immunoprecipitation (ChIP) assays and RNA interference experiments showed that endogenous THAP1 mediates the recruitment of HCF-1 to the RRM1 promoter during endothelial cell proliferation and that HCF-1 is essential for transcriptional activation of RRM1. Together, our findings suggest HCF-1 is an important cofactor for THAP1. Interestingly, our results also provide an unexpected link between DYT6 and DYT3 (X-linked dystonia-parkinsonism) dystonias because the gene encoding the THAP1/DYT6 protein partner OGT maps within the DYT3 critical region on Xq13.1.

Control of α-herpesvirus IE gene expression by HCF-1 coupled chromatin modification activities

The immediate early genes of the alpha-herpesviruses HSV and VZV are transcriptionally regulated by viral and cellular factors in a complex combinatorial manner. Despite this complexity and the apparent redundancy of activators, the expression of the viral IE genes is critically dependent upon the cellular transcriptional coactivator HCF-1. Although the role of HCF-1 had remained elusive, recent studies have demonstrated that the protein is a component of multiple chromatin modification complexes including the Set1/MLL1 histone H3K4 methyltransferases. Studies using model viral promoter-reporter systems as well as analyses of components recruited to the viral genome during the initiation of infection have elucidated the significance of HCF-1 chromatin modification complexes in contributing to the final state of modified histones assembled on the viral IE promoters. Strikingly, the absence of HCF-1 results in the accumulation of nucleosomes bearing repressive marks on the viral IE promoters and silencing of viral gene expression.

Targeting the JMJD2 histone demethylases to epigenetically control herpesvirus infection and reactivation from latency.

Inhibitors of cellular histone demethylases, enzymes required for herpesvirus infection, block early-stage infection and prevent reactivation from latency, demonstrating the potential benefit of epigenetic antiviral therapeutics. Keeping Herpesviruses Under Wraps Despite the pharmaceuticals currently used to control herpesvirus infections and recurrences, herpes simplex virus and its cousin human cytomegalovirus remain important medical pathogens that are responsible for a high incidence of herpetic blindness, complications during organ transplant, and birth defects. In addition, antiherpetic drugs target a late stage in viral infection, allowing drug-resistant viral strains to escape, and resulting in tissue damage from immune-mediated inflammation and subclinical shedding of infectious virus particles. A big goal is to develop drugs that both target the very early events in viral infection and prevent reactivation of the virus from its latent state. Upon infection of a cell with herpes simplex virus or human cytomegalovirus, the cell suppresses the expression of the first class of viral genes by wrapping the viral genome in a type of repressive nucleosomal structure that the cell uses to silence its genes. These viruses, however, have evolved in their ability to commandeer the cellular enzymatic machinery to reverse this repressive packaging, allowing the expression of the viral genes and initiation of productive infection. Identification of the specific enzymes required by these two viruses led Liang et al. to isolate a new inhibitor that blocked the “unwrapping” of the viral genomes. This compound potently suppressed infection of cultured cells with herpes simplex virus or human cytomegalovirus and suppressed the reactivation of herpes simplex virus from latency in a mouse model. Inhibitors such as the compound described in the Liang et al. study represent a new approach to suppressing early events in viral infection that may prevent the rise of resistant viral strains, limit damaging inflammation, and block viral shedding and transmission. Chromatin and the chromatin modulation machinery not only provide a regulatory matrix for enabling cellular functions such as DNA replication and transcription but also regulate the infectious cycles of many DNA viruses. Elucidation of the components and mechanisms involved in this regulation is providing targets for the development of new antiviral therapies. Initiation of infection by herpes simplex virus (HSV) requires the activity of several cellular chromatin modification enzymes including the histone demethylases LSD1 and the family of JMJD2 proteins that promote transcriptional activation of the initial set of viral genes. Depletion of the JMJD2 members or inhibition of their activity with a new drug results in repression of expression of viral immediate early genes and abrogation of infection. This inhibitor also represses the reactivation of HSV from the latent state in sensory neurons. Like HSV, the β-herpesvirus human cytomegalovirus also requires the activity of LSD1 and the JMJD2s to initiate infection, thus demonstrating the potential of this chromatin-based inhibitor to be useful against a variety of different viruses.

Crosstalk between O-GlcNAcylation and proteolytic cleavage regulates the host cell factor-1 maturation pathway

Host Cell Factor 1 (HCF-1) plays critical roles in regulating gene expression in a plethora of physiological processes. HCF-1 is first synthesized as a precursor, and subsequently specifically proteolytically cleaved within a large middle region termed the proteolytic processing domain (PPD). Although the underlying mechanism remains enigmatic, proteolysis of HCF-1 regulates its transcriptional activity and is important for cell cycle progression. Here we report that HCF-1 proteolysis is a regulated process. We demonstrate that a large proportion of the signaling enzyme O-linked-N-acetylglucosaminyl transferase (OGT) is complexed with HCF-1 and this interaction is essential for HCF-1 cleavage. Moreover, HCF-1 is, in turn, required for stabilizing OGT in the nucleus. We provide evidence indicating that OGT regulates HCF-1 cleavage via interaction with and O-GlcNAcylation of the HCF-1 PPD. In contrast, although OGT also interacts with the basic domain in the HCF-1 amino-terminal subunit, neither the interaction nor the O-GlcNAcylation of this region are required for proteolysis. Moreover, we show that OGT-mediated modulation of HCF-1 impacts the expression of the herpes simplex virus immediate-early genes, targets of HCF-1 during the initiation of viral infection. Together the data indicate that O-GlcNAcylation of HCF-1 is a signal for its proteolytic processing and reveal a unique crosstalk between these posttranslational modifications. Additionally, interactions of OGT with multiple HCF-1 domains may indicate that OGT has several functions in association with HCF-1.

Neuronal Stress Pathway Mediating a Histone Methyl/Phospho Switch Is Required for Herpes Simplex Virus Reactivation

Herpes simplex virus (HSV) reactivation from latent neuronal infection requires stimulation of lytic gene expression from promoters associated with repressive heterochromatin. Various neuronal stresses trigger reactivation, but how these stimuli activate silenced promoters remains unknown. We show that a neuronal pathway involving activation of c-Jun N-terminal kinase (JNK), common to many stress responses, is essential for initial HSV gene expression during reactivation. This JNK activation in neurons is mediated by dual leucine zipper kinase (DLK) and JNK-interacting protein 3 (JIP3), which direct JNK toward stress responses instead of other cellular functions. Surprisingly, JNK-mediated viral gene induction occurs independently of histone demethylases that remove repressive lysine modifications. Rather, JNK signaling results in a histone methyl/phospho switch on HSV lytic promoters, a mechanism permitting gene expression in the presence of repressive lysine methylation. JNK is present on viral promoters during reactivation, thereby linking a neuronal-specific stress pathway and HSV reactivation from latency.

Inhibition of LSD1 reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes

Epigenetic suppression of herpes simplex virus demonstrates an approach to control persistent viruses. Epigenetic Control of Herpes Epigenetic modifications are a sort of genetic metadata—they alter gene expression without changing the underlying DNA. Hill et al. hypothesized that epigenetic modification could be a therapeutic approach for treating herpesvirus infection. Indeed, herpesvirus infection and reactivation from latency previously has been shown to depend on the histone demethylases LSD1 and JMJD2. The authors found that blocking the activity of LSD1 could inhibit viral infection in animal models that represent three different stages of herpes simplex virus infection: suppression of primary infection, a block to subclinical shedding, and reduction in recurrent lesions. If these data are confirmed in humans, epi-pharmaceuticals may provide a promising therapeutic avenue to treat this prevalent, lifelong disease. Herpesviruses are highly prevalent and maintain lifelong latent reservoirs, thus posing challenges to the control of herpetic disease despite the availability of antiviral pharmaceuticals that target viral DNA replication. The initiation of herpes simplex virus infection and reactivation from latency is dependent on a transcriptional coactivator complex that contains two required histone demethylases, LSD1 (lysine-specific demethylase 1) and a member of the JMJD2 family (Jumonji C domain–containing protein 2). Inhibition of either of these enzymes results in heterochromatic suppression of the viral genome and blocks infection and reactivation in vitro. We demonstrate that viral infection can be epigenetically suppressed in three animal models of herpes simplex virus infection and disease. Treating animals with the monoamine oxidase inhibitor tranylcypromine to inhibit LSD1 suppressed viral lytic infection, subclinical shedding, and reactivation from latency in vivo. This phenotypic suppression was correlated with enhanced epigenetic suppression of the viral genome and suggests that, even during latency, the chromatin state of the virus is dynamic. Therefore, epi-pharmaceuticals may represent a promising approach to treat herpetic diseases.